The Gene Editing Transduction and Nanotechnology Core has tested and currently offers a variety of lentiviral expression vectors for protein and shRNA expression, and the delivery of transcription factor reporters. These vectors are both commercial and internally acquired from Core collaborators and patrons. The DNA/RNA Delivery Core offers vector maps and sequences, and consults on approaches for cloning in the available vectors. Below you will find a short discussion of the vectors available from the GET iN Core below.
Lentiviral vectors for protein expression:
- pLVX family of vectors (Clontech) which contain CMV promoter that drivers expression of a cDNA, PGK promoter that controls expression of puromycine resistance gene. These vectors contain GFP to be expressed as protein fusion tag on either C or N terminus.
- pCDH family of expression vectors (System Biosciences) are the Core's most popular protein expression vectors due convenient design of their multiple cloning sites and optimized choice of promoters for control of protein expression (CMV) and expression of the antibiotic resistance gene (EF- 1alpha). CMV and EF1alpah promoters are most similar in the expression potential, although EF- 1alpha is slightly weaker than CMV, that provides high expression levels of both transgene and antibiotic resistance gene.
- pLU lentiviral vector (Wistar Institute) implied by the Core for expression of secreted proteins such as cytokines and growth factors. The vector harbors PGK promoter controlling expression of a protein of interest that is linked with RFP through IRES sequence. The PGK promoter provides robust expression in various human and mouse cell types with expression levels close to physiological.
- Protein expression vectors with pre-cloned ORF/cDNA encoding human and mouse proteins, such as pLV (Genecopaeia) and pLOC (Open Biosystems). Being typical lentiviral expression vectors, these are a good choice to acquire vectors that already contain an ORF/cDNA for protein of interest which is ready to be use for virus generation by the Core.
- Vectors for stable and robust cell labeling with fluorescent proteins (GFP, RFP, Tomato) and Luciferase for in vivo experiments on cells injected in to the animals. Vector pFULT vector expresses Luciferase linked to Tamato under the control of the human PGK1 promoter. Vector pGreenFire expresses GFP and Luciferase separated by a T2A link under control of the CMV promoter.
The Core also carries lentiviral expression vectors that express different antibiotic resistance genes such as neomycine, blactocidine and puromycine. Therefore, if available, the Core can offer generation of cell lines and primary cells stably infected with two and three proteins.
Lentiviral vectors for expression of shRNA/miR:
The Core has successfully used shRNA expressing vector from several major companies such as Sigma,Open Biosceinces and Capital Biosciences, that offer self-contained libraries of shRNA clones pre-cloned in their proprietary vectors.
- pGIPZ and its analogue pTRIPZ for inducible expression (Open Biosystems) express RNA under polymerase type II CMV promoter in a form of immature shRNA in the microRNA expressing cassette. These vectors offer tracking of the shRNA expression by GFP and selection of positive clones with puromycine. The pGIPZ library is available at a discounted price through the Northwestern University RNAi/Throughput Core which collaborates with DNA/RNA Delivery Core to generate shRNA expressing viruses for the NU community.
- pLKO family of vectors (Sigma) that offer RNA expression in a form of the immature shRNA under polymerase type III promoter U6. PLKO vector also allow for selection of the infected cells with puromycine.
- pLV vectors (Capital Biosciences) for expression of mature RNAi that does not require post-processing by DICER and DROSHA proteins. These vectors express mature RNAi duplex under U6 and H1 promoters control in a counter direction from 5'and 3' ends of the RNAi sequence. In addition these vectors offer expression of the GFP tracker and an antibiotic of resistance gene. The above bidirectional dual promoter design, allows the user to express and study function of a specific RNAi/miR in cell and animal models that lack expression of DICER and/or DROSHA, and therefore are considered to have no endogenous miRs.
Lentiviral vectors for delivery of transcription factor Luciferase reporters
- Lentiviral expressing vectors delivering DNA binding elements for transcription factors upstream of Luciferase and GFP. These vectors are analogues of well-known Luciferase reporters used throughout for transient transfections of target cells to study activity of transcription factors. To date, the Core has acquired reporters for the following transcription factors: NF-kappaB, AP1, p53, HIF1alpha, TCF/LEF, NOTCH1, Steroid response element, human Involucrin promoter, Endoplasmic stress response element.
Alex Yemelyanov, PhD, 312-503-4192